Th.e long-term goals of this research are to develop robust methods for tracking single proteins in living cells. Recently developed technologies of protein splicing will be used to ligate fluorescent nanocrystals [quantum dot (QD)] derivatives to select proteins in living cells. The availability of such cellular reagents, in combination with modem fluorescence microscopy methods such as to total-internal-reflection microscopy and spectral imaging, will allow insight on protein activity that would be difficult to obtain using macromolecular measurements where protein activities are averaged. There are three specific aims in the proposal: Aim 1" To extend and optimize recently developed in vivo protein trans-splicing and expressed protein ligation approaches to allow the ligation of suitable QD derivatives to either cytosolic and integral membrane proteins. In addition, we will develop a conditional protein trans-splicing approach that will allow probes such as quantum dots to be ligated to proteins following a dcsignatcd functional interaction. This will allow the cellular fate of "activated" proteins to be monitored. Aim 2" To develop methods for preparing monvalent QDs capable of being ligated to a single copy of a target protein in cells. In addition, to develop a strategy, based on fluorescence quenching, that will allow the fluorescence properties of a QD to be linked to the in vivo trans-splicing reaction. This will allow the fluorescence of a protein-QD ligation product to be distinguishable from unreacted QDs. Aim 3: To apply in vivo protein ligation and quantum dots to single proteins in vivo. The technology will be established in the context of two systems of biological interest: exocytosis and transport through the nuclear pore. For each system there is a set of questions that require the tracking of individual proteins. For example, exploring whether protein movement through the pore is primarily diffusive, driven by thermal fluctuations, or deterministic, driven by a molecular motor. Longer-term strategic directions will include trying to develop the technology for chemically tagging interacting proteins and technologies for tracking proteins for longer time periods as they move in three dimensions.